There is accumulating scientific evidence showing that stressors enhance addictive behaviors and are a common cause of relapse to substance abuse. Corticotrophin releasing factor (CRF) is a 41-aa peptide that has been shown to induce various behavioral changes related to adaptation to stress. The CRF system, including the CRF-binding protein (CRF-BP) and the CRF receptors, CRF-R1 and CRF-R2, are thought to contribute, to the physiological adaptations that result from stress. It has also been shown that CRF interaction with CRF-BP may positively modulate CRF-R2 function and, further that when CRF binds to the CRF-BP, it modulates CRF-R2 signaling and contributes to stress-induced relapse to drug seeking. The aim of this application is to identify a chemical series of ligands and compounds that disrupt the interaction between CRF- BP and CRF-R2 to prevent relapse to drug seeking behaviors. Non-peptidyl chemical inhibitors would have advantages over CRF peptides, in terms of cell permeability, stability, and in vivo pharmacology. In the past, this has been difficult due to an inability to develop a suitable high through-put assay for screening against CRF-BP. To address this problem, we have developed an innovative and novel fluorescence based calcium assay where CRF-BP is expressed and tethered at the cell surface in a heterodimeric complex with CRF-R2. This has greatly facilitated our ability to find molecules that inhibit CRF-R2 activation in the presence and absence of CRF-BP. This innovation forms the basis of the high-throughput assay that we have optimized for chemical library screening. We propose to screen a targeted synthetic compound library using this assay and identify chemical inhibitors of CRF-BP-CRF-R2 receptor complex-mediated signaling. Two types of secondary assays will independently confirm any hits. Structure Activity Relations (SAR) and hit to lead optimization will be performed for prototypical inhibitors of the CRF-BP-CRF-R2 receptor complex leading to exploratory pharmacology and preclinical development. Altogether, these efforts will result in validated chemical probes for studying the biology of CRF-BP-CRF-R2 interaction in a variety of cellular and physiological contexts, with the view to developing new therapeutics for treatment of addiction.